WO2016180379A1 - Novel transposon that promotes the functional expression of genes in episomal dna, and method for increasing dna transcription in the functional analysis of metagenomic libraries - Google Patents

Novel transposon that promotes the functional expression of genes in episomal dna, and method for increasing dna transcription in the functional analysis of metagenomic libraries Download PDF

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WO2016180379A1
WO2016180379A1 PCT/CO2015/000010 CO2015000010W WO2016180379A1 WO 2016180379 A1 WO2016180379 A1 WO 2016180379A1 CO 2015000010 W CO2015000010 W CO 2015000010W WO 2016180379 A1 WO2016180379 A1 WO 2016180379A1
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vector
dna
artificial
transposon
promoter
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French (fr)
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Alvaro MONGUI
Patricia DEL PORTILLO OBANDO
Silvia RESTREPO RESTREPO
Armando Junca HOWARD
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Universidad De Los Andes
Corporación Corpogen
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Abstract

A novel transposon (TnC_T7) has been developed to partially supply the transcriptional machinery during the functional analysis of genomic/metagenomic libraries. This transposon was designed and constructed such that it can be integrated randomly into any episomal DNA, allowing the inducible expression of the adjacent DNA regions in both directions. In general, this genetic tool includes a kanamycin resistance gene, two T7 bidirectional promoters and the T7 RNA-polymerase-coding gene, the latter under the control of an arabinose-inducible promoter (PBAD). Experimental validation confirmed the potential of TnC_T7 for use in functional genomic/metagenomic studies in order to partially overcome the limitations of the bacterial hosts, which prevent same from recognising the majority of foreign genes in DNA libraries.

Description

 NEW TRANSPOSON THAT PROMOTES THE FUNCTIONAL EXPRESSION OF GENES IN EPISOMAL DNAs AND A METHOD TO INCREASE THE TRANSCRIPTION OF DNA IN FUNCTIONAL ANALYSIS OF LIBRARIES

METAGENOMICS

Field of the invention

The present invention relates to the development of transposons to partially supply the transcriptional machinery during the functional analysis of genomic / metagenomic libraries, and therefore, to increase the identification of new compounds with biotechnological potential for genomic / metagenomic DNA libraries, surpassing partially the limitations of the bacterial hosts, which prevent them from recognizing most of the foreign genes in the DNA libraries.

BACKGROUND OF THE INVENTION

It is currently estimated that only about one percent of all microorganisms present in natural environments can be grown under standard laboratory conditions, so the great potential of novel compounds, enzymatic activities and genetic regulators useful for the industry is still unknown. , derived from the largest proportion of organisms in the terrestrial biosphere.

In view of the above, that is, due to the limitations to characterize a high proportion of enzymes and metabolites produced by non-culturable bacteria, metagenomics has emerged as an alternative approach to conventional microbiological analysis. This strategy is based on the extraction of total DNA from an environmental sample (genomic mixture of the microbial community known as metagenome) and its subsequent cloning into an easily cultivable bacterium. With this approach, genomic / metagenomic libraries have been constructed and have been used to identify bacterial isolates capable of producing enzymes and novel metabolites. Functional analyzes are then performed based on the heterologous expression of foreign DNA, which is reflected in a particular trait (phenotype)

i expressed by some bacterial isolates from the genomic / metagenomic library.

Therefore, the functional analyzes of most DNA libraries depend on the efficient expression of heterologous genes in the bacterial host to achieve the identification of functions derived from known-unknown genes or complex genetic groups. The success of a given functional assay will largely depend on the detection method and the activity of interest, and three general types of analysis can be distinguished: 1) Direct activity detection, where the expression of a particular enzyme or metabolite is used to identify the bacterial clones; 2) Modulated detection, which involves the expression of genes required for bacterial growth under selective conditions; and 3) Induction by substrate, as a strategy that promotes gene expression in the presence of a given substrate.

However, regardless of the type of detection, functional analyzes are frequently problematic due to the fact that the identification of the desired phenotypes depends on many factors, such as the vector-host system selected, the size of the gene of interest (individual or group of genes), its abundance in the metagenomic source, the detection method used and the efficiency of the heterologous expression of genes in the selected host.

In most of the genomic / metagenomic studies conducted, E. coli has been the preferred host for the construction of libraries and the performance of functional analyzes due in large part to the current understanding of the molecular genetics of this bacterium and its widespread use for decades. as a model in areas such as microbiology and molecular biology. In addition, the development and implementation of sophisticated techniques of genetic modification, together with its simple manipulation, rapid growth, ease of processing and versatility in a wide range of genetic tools, have made E. co // the preferred host in biology experiments molecular.

Despite these advantages, the genetic machinery of E. coli may be unable to correctly perform the gene expression of foreign DNA. The metagenomes are complex mixtures of genomes from a wide range of microorganisms and even a single-genome library could be obtained from a distantly related microorganism. Gabor et al. quantified the theoretical probability of E. coli expressing genes derived from randomly cloned fragments of 32 complete prokaryotic genomes (Env Microbiol 2004, 6: 879-886). This was determined in silico based on the presence of functional signals of E. coli on said genomes and the length of the genomic inserts. Using three theoretical models of gene expression, it was found that on average 40% of the enzymatic activities encoded by prokaryotic genomes could be accessible to the machinery of E. coli. This means that a significant portion of genes from these genomes (60%) would still be incompatible for the transcriptional and translational machinery of the bacterial expression system.

A more recent study to determine the ability of E. coli to globally transcribe different genes, both prokaryotes and eukaryotes, was evaluated by microarrays and RT-PCR (Warren RL, et al., Genome Res. 2008, 18: 1798-1805) . It was observed that E. coli was capable of transcribing about half of the Haemophilus influenzae genes, a smaller proportion of P. aeruginosa genes and only a minimal number of human genes. Additionally, the genes that showed significantly higher levels of expression in E. coli had promoter regions related to the recognition sites of the sigma-70 subunit of bacterial RNA polymerase, highlighting the selectivity of the transcriptional machinery of the host during the first steps of the expression of foreign DNA.

Therefore, the selectivity of the transcriptional machinery is evident during the initial stages of foreign DNA expression, which implies that any strategy aimed at increasing gene expression in functional genomic / metagenomic studies, independent of the selected bacterial host, must overcome this initial limitation.

Different strategies have been reported to improve the heterologous expression of genomic / metagenomic DNA genes. So, for example, the use of Alternative hosts, whether the DNA library is constructed simultaneously in some of them or that is transferred from one host to another, have been shown to be successful in increasing gene expression. This strategy is usually associated with the development of novel expression vectors that can be stably maintained in more than one bacterial system.

An example of development (but not functional implementation) of host broad-spectrum vectors or shuttle vectors was pSR44 (disclosed by Aakvik T. et al, FEMS Microbiol Lett 2009, 296: 149-58). This vector can be induced from a low number of copies up to a high number of copies with L-arabinose and contains a transfer origin RK2 to allow conjugation in additional hosts such as Pseudomonas fluorescens and Xanthomonas campestris. Although in recent years the repertoire of shuttle vectors has increased, still few studies have confirmed the versatility of these genetic tools to increase the heterologous expression of genes in functional analysis of metagenomic libraries.

Another modification in vectors used for the construction of genomic / metagenomic libraries is the addition of promoters adjacent to the multiple cloning sites in order to increase the transcription of the foreign DNA. For obvious reasons, the effectiveness of this strategy is more restricted to libraries built with small DNA inserts. This is the case of plasmid pJOE930, which having two convergent and inducible lac-promoters on both sides of a multiple cloning symmetric site allowed the identification of a large number of active bacterial isolates, expressing lipolytic enzymes, amylases, phosphatases and dioxygenases ( Lámmle K, et al., J. Biotechnol., 2007, 127: 575-92).

The patent application WO 2012/069668 is related to the development of vectors and strains as expression systems, offering the possibility of identifying genes of interest that are not expressed in bacteria that host the metagenomic library, and thus allowing the detection of the encoded functions, which, otherwise, would remain silenced and could not be detected. Specifically, said patent application reveals the inclusion of the promoter derived from the T7 phage in cosmid and fosmido vectors to promote the transcription of genomic / metagenomic inserts as a result of the expression of the T7RNA polymerase (T7RNAP) from the host. The success of this strategy is based on the high processivity and efficiency of the T7RNAP for transcribing genes, but it is restricted to the analysis of the flanking regions of the genomic / metagenomic inserts.

Another approach is the use of mobile DNA elements or transposons, which have been widely used in a variety of advanced genetic studies, such as mutagenesis, sequencing (US2014 / 0162897), genomic manipulation, transgenesis, gene therapy and functional modulation of the gene expression (Ivics Z. et al .; Nal Methods 2009, 6: 415-22).

One of the best characterized transposition machinery is the bacteriophage Mu, since in contrast to the relative complexity of the in vivo transposition mechanism of this phage, which involves a significant number of auxiliary factors, substantially fewer conditions have been observed for the reactions of in vitro transposition. Thus, the minimal reaction components for Mu-type transposition include the reaction buffer, the purified MuA transposase, the Mu mini transposon, and the DNA of interest (target DNA). These parameters have been shown to be sufficient for efficient transposition events with low insertion bias over multiple target DNAs, making the implementation of the Mu transposon an ideal and adjustable tool in different fields of research.

In terms of applications in molecular biology, the Mu transposon has facilitated sequencing analysis, the detection of polymorphisms and the precise determination of protein interactions. In the field of protein engineering, the Mu transposon has been basically used to generate truncated proteins in order to characterize differential enzymatic activities. At the genomic level, the Mu transposons have widely promoted mutagenesis and transgenesis events, focused on decreasing or increasing the functional expression of genes, respectively, in different organisms. Specifically, Leggewie C. et al. (J. Biotechnol 2006, 123: 281-7) reveal the construction of the MuExpress transposon that is randomly integrated in vitro in existing libraries of bacterial artificial chromosomes (BACs) or in cosmid libraries, favoring the inducible expression of its flanking regions in both directions and allowing bidirectional sequencing of the respective clones from unique binding sites of the primers.

Said MuExpress transposon was developed as a genetic tool to address the difficulty of gene transcription within long DNA inserts of metagenomic libraries. Theoretically, this transposon increases the level of transcription of DNA inserts because it includes, at each of its ends, a T7 promoter region for reading to the outside. However, a detailed analysis of the original design and construction of the MuExpress transposon revealed an important error that makes it impossible to recognize one of the two T7 promoter regions by the T7RNAP.

Another transposon known to randomly insert a single T7 promoter, but derived from the Tn5 transposition system, is EZ-Tn5 <T7 / KAN-2> (Epicenter-Illumina, Madison, WI, USA). However, neither the MuExpress nor the EZ-Tn5 <T7 / KAN-2>, which are based on the high transcription processivity of the T7RNAP, reported sufficient evidence of insertion of the transposons nor their relationship with the improvement in the expression gene Additionally, MuExpress and EZ-Tn5 <T7 / KAN-2> transposons depend on bacterial hosts that express T7RNAP (for example E. coli BL21 DE3, Invitrogen), which greatly restricts their use in functional assays, especially with metagenomic DNA , since most of the library construction kits depend on other specialized bacterial strains (eg E. coli Epi300, Epicenter-Illumina, Madison, Wl, USA).

Based on the versatility of Mu transposons in molecular biology-biotechnology research, as well as the current need to efficiently improve the heterologous gene expression of genomic / metagenomic DNA libraries, it was necessary to create new genetic tools and strategies using This mobile DNA element. Summary of the invention

The present invention involves the design of a new Mu transposon and the methods to achieve efficient expression of genes housed in episomal DNAs of genomic / metagenomic libraries, which under traditional analysis approaches are not detected in functional assays. The efficiency in the use of the invention is reflected in an increased proportion of bacterial isolates that show the desired phenotype, compared to the proportion of bacterial isolates that can be identified in the original functional analyzes. The first aspect of the invention is based on the sequential development of plasmids for the construction of the new Mu transposon.

In one embodiment, the invention is directed to the development of a synthetic gene (Tn_A), which is an artificial DNA sequence resulting from the specific combination of certain DNA elements, comprising:

(i) a promoter sequence T7,

(ii) an inverted repeat recognition site for the transposase

MuA,

(iii) multiple flanking recognition sites for restriction endonucleases.

In another embodiment, the invention is directed to the development of plasmid pUC57_Tn, which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:

(i) a skeleton vector with a high copy number origin of replication and a selection marker,

(ii) a T7 promoter sequence in a specific orientation,

(iii) an inverted repeat recognition site for the MuA transposase in a specific orientation. In another embodiment, the invention is directed to the development of the plasmid pUC57_Tn_kanAB, which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:

(i) a skeleton vector with a high copy number origin of replication and a selection marker,

(I) a T7 promoter sequence of reading outwards,

(iii) an inverted repeat recognition site for the transposase

MuA,

(iv) a selection marker different from that which is located in the skeleton vector.

In another embodiment, the invention is directed to the development of plasmid pBAD18-Cm_t7rnap, which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:

(i) a skeleton vector with a high copy number origin of replication and a selection marker,

(ii) a gene encoding the T7RNA polymerase regulated by an inducible promoter.

In another embodiment, the invention is directed to the development of the plasmid pUC57_Tn_kanAB_t7, which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:

(i) a skeleton vector with a high copy number origin of replication and a selection marker,

(ii) a T7 promoter sequence reading to the outside,

(iii) an inverted repeat recognition site for the transposase

MuA,

(iv) a selection marker different from that which is located in the skeleton vector, (v) a gene encoding the T7RNA polymerase regulated by an inducible promoter.

In another embodiment, the invention is directed to the development of plasmid pUC57_TnC_T7 which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:

(i) a skeleton vector with a high copy number origin of replication and a selection marker,

(ii) a selection marker different from that located in the skeleton vector,

(iii) a gene that encodes the T7RNA polymerase under an inducible promoter,

(iv) two T7 flanking promoters reading to the outside,

(v) two inverted repeat recognition flanking sites for the MuA transposase.

In another embodiment, the invention is directed to the development of F076 GFP phosphide which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:

(i) a skeletal fosmoid with low to high copy number origins of replication and a selection marker,

(¡) A metagenomic DNA insert,

(iii) a gene encoding a variant of the green fluorescent protein (GFP) with a ribosome binding site (RBS) upstream, both specifically located within the metagenomic DNA insert.

In another embodiment, the present invention is broadly directed to a method for increasing DNA transcription, as an initial step for foreign gene expression, comprising:

(i) generate episomal DNA transposition libraries, as a result of the random insertion of the purified TnC_T7 transposon, where said episomal DNA includes plasmids, fosmides, cosmids or BACs, (i) introduce the episomal DNA transposition libraries of (i) within host cells,

(iii) expressing the T7RNA polymerase encoded in the TnC_T7 transposon, to provide the populations of bacterial host cells with a diverse collection of RNA transcripts derived from episomal DNA,

(iv) analyzing said population of bacterial host cells to identify bacterial isolates that express a reporter gene or any other desired function.

In another embodiment, the present invention includes eight plasmids, which correspond to artificial vectors resulting from random insertions of the TnC_T7 transposon in the plasmid pKR-C12, each comprising:

(i) a skeleton vector with its own selection marker and a silenced reporter gene,

(ii) a differential insertion of the TnC_T7 transposon along the

 DNA sequence of (i).

Brief description of the figures

The following figures are part of the present description and are included to additionally demonstrate certain aspects thereof. The invention can be better understood by reference to one or more of these figures, in combination with the detailed description of the specific embodiments presented herein.

FIG. 1 shows the structure of the Tn_A DNA sequence, which comprises the binding sequence R1 -R2 for the MuA transposase, corresponding to one of the Inverse repeat recognition sites, and to the T7 promoter region in the same DNA molecule, but in the opposite strand. The length of the Tn_A gene is 138 bp and includes flanking recognition sites for the following restriction endonucleases: EcoRI, BglW, Asc and 8amHI. FIG. 2 outlines the cloning of the Tn_A DNA sequence in the vector pUC57 to generate the plasmid pUC57_Tn. Direct cloning was achieved using the EcoRI and Bam restriction sites in the gene and in the plasmid pUC57, as a requirement for subsequent steps in order to construct the plasmid harboring the TnC_T7 transposon. AmpR, ampicillin resistance gene; M13 fwd, hybridization site for the direct M13 primer; M13 rev, hybridization site for the reverse M13 primer; ori, origin of replication ColE1 / pMB1 / pBR322 / pUC high number of copies; CAP binding site, catabolite activator protein binding site.

FIG. 3 describes the cloning in pUC57_Tn of the kanamycin resistance gene (Kan) including its promoter, to generate the plasmid pUC57_Tn_kanAB. First Kan was amplified in two steps, in order to replace the BglW restriction site with Spel. The ligated product was inserted into the restriction sites Asc \ and BamH \ of pUC57_Tn. AmpR, ampicillin resistance gene; M13 fwd, hybridization site for the direct M13 primer; M13 rev, hybridization site for the reverse M13 primer; ori, origin of replication ColE1 / pMB1 / pBR322 / pUC high number of copies; CAP binding site, catabolite activator protein binding site; NeoR / KanR, neomycin and kanamycin resistance gene; FRT, excision site but not integration mediated by FLP.

FIG. 4 shows the cloning of the sequence coding for the T7RNA polymerase (T7RNAP) at the unique restriction site Kpn \ (after the end of the repair) of the plasmid pBAD18-Cm (Guzmán LM, et al., J. Bacterio !. 1995, 177: 4121-30), to generate the plasmid pBAD18-Cm_t7rna. The coding sequence of T7RNAP is located downstream of the arabinose-inducible promoter (PBAD) and upstream of the transcriptional terminators rrnB T1 and T2. CmR, chloramphenicol resistance gene; f1 ori, origin of replication of bacteriophage f1; or, high copy number ColE1 / pMB1 / pBR322 / pUC origin of replication; bom, base of the mobility region for pBR322; araC, regulatory protein of L-arabinose; araBAD promoter, promoter of the E. coli L-arabinose operon. FIG. 5 represents the detection of the AA peptide in a western blot assay, as a result of the expression of T7RNAP derived from different extracts of bacterial cultures. 1, positive control of the expression of the AA peptide from the bacterial clone E. coli BL21 DE3 transformed with the plasmid pET28a_AA and supplemented with kanamycin and IPTG; 2, bacterial clone E. coli TOP10 transformed with the plasmids pET28a_AA and pBAD18-Cm_t7rnap and supplemented with kanamycin, chloramphenicol and D-glucose; 3-6, E. coli TOP10 bacterial clones transformed with the plasmids pET28a_AA and pBAD18-Cm_t7rnap and supplemented with kanamycin, chloramphenicol and L-arabinose.

FIG. 6 outlines the cloning of the arabinose-inducible promoter and the coding sequence of T7RNAP, as a single amplicon, at the unique restriction site> Ascl of pUC57_Tn_kanAB, generating the plasmid pUC57_Tn_kanAB_t7. The P B AD_T7RNAP amplicon is located between the Tn_A DNA sequences and the kanamycin resistance gene. AmpR, ampicillin resistance gene; M13 fwd, hybridization site for the direct M13 primer; M13 rev, hybridization site for the reverse M13 primer; ori, origin of replication ColE1 / pMB1 / pBR322 / pUC high number of copies; CAP binding site, catabolite activator protein binding site; NeoR / KanR, neomycin and kanamycin resistance gene; FRT, excision site but not integration mediated by FLP; T7RNAP, coding sequence of the T7RNA polymerase; araBAD promoter, promoter of the L-arabinose operon of E. coli.

FIG. 7 outlines the cloning of the second end of the transposon (Tn_B) in pUC57_Tn_kanAB_t7 to generate the plasmid pUC57_TnC_T7. Tn_B included the other binding site R1-R2 for the MuA transposase, the second T7 promoter region of the final construction of the TnC_T7 transposon, and two Hind restriction frameworks \\\, which allow its cloning into the target vector. The BglW restriction sites that are necessary to release the TnC_T7 transposon from the plasmid are also highlighted. AmpR, ampicillin resistance gene; M13 fwd, hybridization site for the direct M13 primer; M13 rev, hybridization site for the reverse M13 primer; ori, origin of replication ColE1 / pMB1 / pBR322 / pUC high number of copies; site of union CAP, site of catabolite activator protein binding; NeoR / KanR, neomycin and kanamycin resistance gene; FRT, excision site but not integration mediated by FLP; T7RNAP, coding sequence of the T7RNA polymerase; araBAD promoter, promoter of the E. coli L-arabinose operon.

FIG. 8 represents to scale the structural regions of the transposon TnC_T7. Each of the two binding sites for the MuA transposase is adjacent to a single T7 promoter. In italics the most representative restriction sites in the construction of the transposon are highlighted. The distance between the two BglU restriction sites is 4,575 bp. KanR, kanamycin resistance gene; araBAD promoter (arabinose inducible promoter (PBAD)), promoter of the E. coli L-arabinose operon; T7RNAP, coding sequence of the T7RNA polymerase.

FIG. 9 represents the principle of the method disclosed here, aimed at increasing the transcription of DNA as the initial step of the expression of foreign genes. Specifically, the method comprises the use of the TnC_T7 transposon, a Mu transposon, to partially supply the transcriptional machinery during functional analyzes of genomic / metagenomic libraries. This transposon was conceived and constructed to have the ability to integrate randomly into any episomal DNA, allowing inducible expression of adjacent regions of DNA in both directions. A, B and C show examples of transposition events of TnC_T7 on white DNA and the ability in each case to increase gene expression as a result of its specific insertion. In case there is a gene of interest (black boxes with white arrows showing the reading orientation) in a particular white DNA, the random insertion of the TnC_T7 transposon can promote DNA transcription (dotted arrows), which will eventually lead to the expression of particular proteins (black circles) and the detection of the desired phenotype.

FIG. 10 represents the initial detection of .bacterial clones expressing GFP, based on the random insertion of the TnC_T7 transposon into pKR-C12 (1-15). The detection of fluorescence was performed by spectrophotometry and expressed in terms of Relative Fluorescence Units (RFUs). As Negative (-) and positive (+) fluorescence controls were incubated E. coli Epi300 pKR-C12 in the absence and presence of A / - (3-oxododecanoyl) -L-homoserine lactone (3-oxo-C12-HSL) 5 μΜ (Sigma-Aldrich, Saint Louis, MO, USA), respectively.

FIG. 1 1 outlines the insertions of the TnC_T7 transposon in pKR-C12 and the fluorescence detection of selected posttransposition bacterial isolates. A, scale diagram of plasmid pKR-C12. The fragment of the highlighted plasmid shows the exact location of the transposon insertions (associated with bacterial clones 1-8) located in the vicinity of the gene encoding GFPmut3 * (GFP). The dotted arrows pointing from top to bottom represent the transposon insertion sites in which the t7rnap gene is located in the sense strand of the target DNA, while the bottom-up arrows represent the same gene located in the antisense strand of the DNA . pBBR1, origin of replication of the plasmid; LasR, transcriptional regulator; PO, Lac promoter and Lac operator system; GFP, green fluorescent protein; CmR, chloramphenicol resistance gene; GmR gene for resistance to gentamicin; CAP, catabolite activator protein binding site; * , two independent insertions of TnC_T7 in the same position. B, fluorescence detection by spectrophotometry expressed in terms of RFUs / Optical Density6oonm (GFP / OD); 1-8, isolated from E. coli Epi300 harboring the plasmids with the transposon inserts shown in A; (-) Negative control of GFP expression; (+) positive control of GFP expression. C, fluorescence detection by the IVIS Image System detector; 1-8, isolated from E. coli Epi300 harboring the plasmids with the transposon inserts shown in A.

FIG. 12 represents a fosmido derived from pCC2FOS, which includes a metagenomic DNA insert of soil (F076) and wherein the coding sequence of GFPmut3 * was cloned, to generate the F076_GFP fosmid. The restriction site AscI was used for the cloning of the coding sequence of GFPmut3 * into the metagenomic insert of the F076 phosphide. M13 fwd, hybridization site for the direct M13 primer; M13 rev, hybridization site for the reverse M13 primer; CAP binding site, protein binding site of catabolite activator; CmR, chloramphenicol resistance gene; oriV, origin of replication for the bacterial F plasmid; ori2, secondary origin of replication for the bacterial F plasmid; repE, replication initiation protein for the bacterial plasmid F; incC, region of incompatibility of the bacterial F plasmid; sopA and B, partition proteins of the bacterial F plasmid; sopC, partition region similar to the centromere of the bacterial F plasmid; loxP, recombination site mediated by Cre.

FIG. 13 outlines the detection of fluorescence of bacterial isolates of E. coli transformed with F076_GFP after transposition with TnC_T7 and the location of the transposon insertions along the fosmido sequence. A, fluorescence detection using the IVIS Imaging System for E. coli Epi300 isolates transformed with the transposition reaction of TnC_T7 in F076_GFPmut3 *. Negative (-) and positive (+) controls of GFP expression were included, corresponding to bacterial isolates 2 and 4 of E. coli Epi300, respectively, of FIG. 1 1 C. Background basal fluorescence was corrected based on the auto fluorescence signal of the negative control used. B, revalidation of GFP expression for bacterial isolates harboring the F076_GFPmut3 * fosmido, using the IVIS Imaging System after recovery of the isolates on LB agar with 20 pg / mL chloramphenicol, 40 pg / mL kanamycin and L-arabinose 0.2%. C, scale diagram of F076_GFPmut3 * with the identified transposon insertion sites TnC_T7. GFP, coding sequence of the green fluorescent protein. The boxes and white arrows represent the genes and regulatory sequences of the fosmídico skeleton pCC2FOS. The small segmented arrows oriented from top to bottom represent the transposon insertion sites in which the t7rnap gene is located in the sense strand of the target DNA, while the bottom-up arrows represent the same gene located in the antisense strand of the transposon. DNA The sequence of the metagenomic DNA insert (MI) is highlighted, as well as the ORFs with length greater than 150 codons located in the sense strand of the fosmídico DNA. The shaded box includes the TnC_T7 inserts that promoted the GFP expression. CmR, chloramphenicol resistance gene. FIG. 14 outlines the degradation of 4-nitrophenyl butyrate from the extracts of bacterial clones. BL21, bacterial clone E. coli BL21 DE3 used as negative control; LipN, bacterial clone E. coli BL21 transformed with the plasmid pET100_LipN (bacterial clone kindly provided by Luis Peña - Molecular Biotechnology, CorpoGen, Bogotá, Colombia) used as a positive control; 14gF2, E. coli Epi300 transformed with the fosmido pCC2FOS_14gF2; 1-15, bacterial isolates of Epi300 coli transformed with the pCC2FOS_14gF2 fosmido after transposition with TnC_T7.

Detailed description of the invention

Definitions

Unless defined or otherwise specifically described elsewhere in this text, the following terms and descriptions related to the invention should be understood as described below.

As used herein, "artificial DNA" means a DNA sequence different from any found in nature or produced by an unnatural process, as a result of in vitro techniques or solid phase DNA synthesis.

As used herein, "synthetic gene" means a DNA fragment synthesized in the laboratory by the combination of nucleotides, without pre-existing DNA sequences. In particular, the term refers to a fully synthetic double-stranded DNA molecule.

As used herein, "recognition sites" refers to locations on a DNA molecule that contain specific nucleotide sequences, which are recognized by specific proteins or enzymes.

As used herein, "restriction endonucleases" means enzymes that cut double-stranded DNA molecules at specific recognition sites. The term referred to in the present invention relates to restriction endonucleases or enzymes that specifically recognize DNA sequences of 6-8 nucleotides, in the which the nucleotide sequence of one of the strands of the DNA is read in reverse order to that of the strand of complementary DNA (palindromic).

As used herein, "transposable element" or "transposon" refers to a DNA sequence or a gene segment capable of moving from a genome or genetic position to be inserted in another position (eg, another genome, chromosome , Episomal DNA). The aforementioned definition includes only transposable elements or transposons that are based on intermediate DNA molecules and that require the enzymatic activity of particular proteins, termed transposases, to move along different genetic positions.

As used herein, "transposition" or "transposition reaction" refers to the reaction in which the transposon is inserted into a target DNA at random sites, through the catalytic activity of a transposase.

As used herein, "inverted repeat" means a sequence identified at the 5 'or 3' ends of the transposons that are specifically recognized by transposases.

As used herein, "transposase" is intended to mean an enzyme that has the ability to recognize and bind to one end of a transposon or to the final sequences of a transposon in a transposition reaction, to promote transposon mobilization.

As used herein, "transposon insertions" means specific locations where the transposon is inserted into a target DNA, as a result of the transposition reaction performed by a specific transposase.

As used herein, "white DNA" or "white vector" means a double-stranded DNA that is suitable for modification using molecular biology techniques. In this invention, the definition is associated with episomal DNA sequences that include specific recognition sites for restriction endonucleases or that can be modified by transposases as a result of the inclusion of transposable DNA elements.

As used herein, "DNA transcription" means the process of synthesizing a copy of RNA from a DNA molecule. This corresponds to the first step of gene expression and is carried out by a specialized enzyme, an RNA polymerase.

As used herein, "promoter" refers to a region in a DNA sequence over which a specific RNA polymerase can bind (for example, the T7 promoter is recognized only by T7RNAP), in order to begin the process of transcription of DNA.

As used herein, "inducible promoter" means that the recognition of the promoter by the RNA polymerase and therefore the transcriptional activity can be controlled by the absence or presence of chemical or physical factors. For purposes of the present patent, if a promoter is induced by a specific factor this will lead to the synthesis of a specific protein (for example T7RNAP).

As used herein, "constitutive gene" or "constitutively expressed gene" means a gene that is continuously transcribed at a relatively constant level. This term implies that a constitutive promoter regulates the transcription of DNA for the gene (for example the kanamycin resistance gene) and therefore the constant expression of the resulting protein.

As used herein, "outward reading" refers to the direction of DNA transcription from a specific promoter, which is located particularly within a defined DNA sequence, (such as a transposon) and which is located at the 5 'or 3' ends of the DNA segment. In this case, the reading towards the outside is restricted to the process of DNA transcription from the mentioned promoter, in which RNA synthesis starts from the transposon, but extends mainly towards the DNA adjacent to it. As used herein, "vector" refers to a circular double-stranded DNA molecule used as a vehicle to artificially transport foreign DNA into a target bacterial cell.

As used herein, "artificial vector" refers to any artificial DNA as a vector, which is capable of self-replicating within a bacterial cell and therefore being stably maintained within the host bacterial cell.

As used herein, "auto replication" and "episomal" refer to the ability of a vector or artificial vector not to be integrated into the genomic DNA of a certain cellular host, but to automatically replicate in a cell host and, therefore, be present when the host cell grows and divides. In particular, this term assumes the permanence of the vector or artificial vector for several generations of growth within the host cell. This term includes plasmids, fosmides, cosmids and artificial bacterial chromosomes (BACs).

As used herein, "origin of replication" refers to particular sequences in episomal DNAs in which replication is initiated, based on the recruitment of proteins involved in DNA replication.

As used herein, "transformation" means the process of introducing new genetic material specifically into bacterial cells. In the present invention, the mentioned term is associated with the introduction of vectors, artificial vectors or modified artificial vectors within bacterial cells.

As used herein, "selection marker" refers to a gene located within the bacterium (at the genomic or episomal level) that confers a characteristic for artificial selection. This term is associated with antibiotic resistance genes (for example, the resistance to chloramphenicol) localized in vectors or artificial vectors for the selection of bacterial isolates after transformation.

As used in the present specification, the terms "upstream" and "downstream" are used to differentiate relative positions in DNA or RNA sequences. The upstream position is a position toward the 5 'end of another nucleic acid segment (e.g., a promoter, gene, restriction site, etc.) in a single strand of DNA or in an RNA molecule. The downstream position is a position towards the 3 'of another segment of nucleic acid in a single strand of DNA or in an RNA molecule.

As used herein, "metagenomic DNA" refers to the totality of genomic DNA associated with microbes, isolated from complex samples such as open natural environments (eg soil, water) or from microbiomes of multicellular organisms (e.g. humans).

As used herein, "insert" or "DNA insert" means a part or fragment or sequence of DNA that is inserted by molecular biology techniques into a vector or an artificial vector for subsequent selection, manipulation or expression within a host organism.

As used herein, "ribosome binding site" refers to an RNA sequence in which ribosomes can be joined to initiate the process of protein synthesis (translation) within the host cell or organism, such as part of the process of protein expression.

As used herein, "foreign gene expression" means the entire process by which the information of a particular gene is used to synthesize a product, which for the purposes of the present invention means to synthesize a protein. In the aforementioned term, "foreign" means that the evaluated gene belongs to an organism different from that used to promote the expression of the gene. As used herein, "reporter gene" means a gene whose expression in a bacterial host can be easily monitored or detected. In the context of the present invention, the reporter gene encodes a variant of the green fluorescent protein (GFP).

As used herein, "silenced gene" refers to a gene that is unable to express the protein associated with its coding sequence, either by impediments during the process of transcription or translation within the host cell.

An embodiment of the invention disclosed herein is directed to the design and development of a synthetic gene (Tn_A), which is an artificial sequence of double-stranded DNA of 138 base pairs (bp) resulting from the specific combination of certain DNA elements . This DNA fragment includes a MuA binding site corresponding to the inverted repeat recognition site for the transposase, a T7 promoter sequence that allows the specific interaction of the T7RNAP and the following recognition flanking sites for restriction endonucleases: EcoRI, BglW, Asc \ and SamHI. The aforementioned synthetic gene design was thought to locate the MuA binding site and the T7 promoter in different DNA strands (FIG 1), in order to achieve the desired activity in the final construction of the transposon.

In one embodiment, the present invention includes the plasmid pUC57_Tn, which corresponds to an artificial 2,795 bp vector that can be maintained within a bacterial host cell and wherein said plasmid is the result of a specific combination of certain DNA elements (FIG. ). pUC57_Tn has a skeletal vector with the origin of replication ColE1 / pMB1 / pBR322 / pUC and an ampicillin resistance gene as a selection marker after its transformation into the host cell. pUC57_Tn is the result of cloning the synthetic gene Tn_A in the unique EcoRI and SamHI sites, after a treatment with restriction enzymes of the skeleton vector and the synthetic gene. Therefore, vector pUC57_Tn in the present embodiment includes a T7 promoter reading to the outside and an inverted repeat recognition site for the MuA transposase, both specifically located. One embodiment of the invention includes the plasmid pUC57_Tn_kanAB, which is an artificial vector of a total length of 3.983 bp that can be replicated episomally in a bacterial host cell. Plasmid pUC57_Tn_kanAB results from the combination of certain DNA elements (FIG 3), since it includes the aforementioned DNA regions of pUC57_Tn plus an additional resistance gene. In the present invention, the plasmid pUC57_Tn_kanAB is the result of the cloning of the kanamycin resistance gene, including its promoter and its transcription termination signal at the Asc \ and BamH \ restriction sites of pUC57_Tn. For the present embodiment, the cloning of the kanamycin resistance gene was carried out in a specific manner in order to ensure adequate addition of the subsequent DNA regions.

In one embodiment, the present invention is directed to the development of the plasmid pBAD18-Cm_t7rnap (FIG 4), which is an artificial vector that can be stably maintained within a bacterial host cell. This vector has a total size of 8.738 bp and is the result of the combination of the following DNA elements: origin of replication ColE1 / pMB1 / pBR322 / pUC, a chloramphenicol resistance gene and the coding sequence of the T7RNAP cloned in the unique restriction site Kpn \. Additionally, pBAD18-Cm_t7rnap described in this invention has the coding sequence of T7RNAP located downstream of the arabinose-inducible promoter and upstream of the rrnB transcriptional terminators T1 and T2.

An embodiment of the invention relates to the plasmid pUC57_Tn_kanAB_t7, which is an artificial vector of 7.097 bp (FIG 6), resulting from the specific combination and orientation of certain DNA elements and which can be maintained within a bacterial host cell. Plasmid pUC57_Tn_kanAB_t7 includes the aforementioned DNA elements of pUC57_Tn_kanAB, which are a ColE1 / pMB1 / pBR322 / pUC origin of replication, an ampicillin resistance gene, the Tn_A synthetic gene and the kanamycin resistance gene, plus the inducible promoter with arabinose and the coding sequence of the T7RNAP of vector pBAD18-Cm_t7rnap. The artificial vector pUC57_Tn_kanAB_t7 results specifically from cloning of the arabinose-inducible promoter and the coding sequence of the T7RNAP at the unique restriction site Asc of pUC57_Tn_kanAB, between the DNA sequences of Tn_A and of kanamycin resistance in the artificial vector.

An embodiment of the invention disclosed herein is directed to the development of the plasmid pUC57_TnC_T7 (FIG 7), which is an artificial vector resulting from the specific combination of certain DNA elements and which can be maintained in a bacterial host cell. This artificial vector includes all the structural elements of the vector pUC57_Tn_kanAB_t7 plus the second end of the transposon, denoted here as Tn_B. Plasmid pUC57_TnC_T7 has a total length of 7.240 bp and has specifically cloned the Tn_B region at the unique restriction site Hind \\\ of pUC57_Tn_kanAB_t7. As a consequence, the artificial vector pUC57_TnC_T7 has two outward reading flanking T7 promoters, as well as two inverted repeat recognition sites for the MuA transposase. Therefore, pUC57_TnC_T7 has cloned the complete transposon sequence TnC_T7, which in turn can be released by means of the BglW restriction enzyme treatment.

In one embodiment, the present invention relates to the development of the F076_GFP fosmido (FIG.1 1), which is an artificial vector comprising 45,619 bp. This vector results from combining in a specific manner a phosphid skeleton, a metagenomic DNA insert and the coding sequence of a reporter gene. The phosphine skeleton corresponds to the commercial vector pCC2FOS (Epicenter-Illumina, Madison, WI, USA). The metagenomic insert results from the random cloning of metagenomic DNA in pCC2FOS. The reporter gene corresponds to the GFP coding sequence including a ribosome binding site (RBS), both DNA fragments being cloned as a single amplicon at a unique Asc restriction site located in the metagenomic insert.

One embodiment of the disclosed invention is a method for increasing DNA transcription, including, but not limited to, the expression of foreign genes (FIG 9), which comprises: (i) Generate DNA libraries based on random transposition or on transposon insertions in episomal DNA. Transposition-based DNA libraries can be obtained from purified sequences of episomal DNA, such as plasmids, fosmides, cosmids or BACs, having unique DNA inserts or from sets of episomal DNA sequences each having an insert of Different DNA

(ii) Introducing one or more of said episomal DNA libraries based on transposition of (i) into bacterial host cells by standard transformation methods.

(iii) Induce the expression of the T7RNA polymerase in the resulting bacterial isolates transformed with episomal DNA sequences based on transposition. The specific expression of the T7RNA polymerase from each insertion of the TnC_T7 transposon provides a diverse collection of DNA transcripts or RNA sequences in the resulting bacterial cell population.

(iv) Analyze said population of bacterial host cells to identify specific bacterial isolates that express, but not be limited to, a reporter gene encoding GFP. The expression of the reporter gene, like any other type of phenotype under study using the method disclosed in the present invention, is associated with the specific RNA sequences generated in the analyzed bacterium, which in turn correlates with transposon-specific insertions TnC_T7 in the original episomal DNA libraries.

In one embodiment, the present invention includes eight plasmids corresponding to artificial vectors that result from random insertions of the transposon into pKR-C12, this being a plasmid that includes a silenced reporter gene encoding GFP. The plasmids included in this embodiment are characterized by having a differential TnC_T7 transposon inserted into the original white plasmid and having the same total length. The specific location of the transposon insertion in each case defines the efficiency of the respective bacterial isolate transformed to express the reporter gene.

Examples

Example 1: Inducible expression of T7RNAP from the PBAD promoter

An artificial vector for the recombinant expression of T7RNAP was generated by cloning the coding sequence of the mentioned protein at the multiple cloning site of the plasmid pBAD18-Cm. For this, the coding sequence of the T7RNAP was amplified with Accuzyme as a high fidelity polymerase (Bioline, London, United Kingdom), using as a template purified genomic DNA of the BL21 strain of E. coli (Invitrogen-Life Technologies, Carisbad, CA , USA) and the primers provided in Seq-ID1 and Seq-ID2. On the other hand, vector pBAD18-Cm (Guzman LM, et al., J. Bacteriol., 1995, 177: 4121-30) was linearized by enzymatic restriction with Kpn \ and its DNA ends repaired with T4 DNA polymerase (New England Biolabs, Ipswich, MA, USA). After purification of the PCR amplicon and the vector, a ligation and transformation reaction was carried out in E. coli TOP10 (Invitrogen-Life Technologies, Carisbad, CA, USA), according to standard methods known in the art. art. The correct orientation of the insert was verified by digestion with restriction enzymes, colony PCR and / or sequencing of the plasmid DNA isolated from the resulting bacterial clones (FIG 4).

In a next step, a clone of E. coli TOP10 harboring pBAD18-Cm_t7rnap was transformed with a plasmid that includes the sequence encoding the AA peptide located downstream of a T7 promoter (pET28a_AA). The selection of the resulting bacterial isolates including both plasmids was performed using the corresponding selection markers for both vectors.

To evaluate the expression of the coding sequence of the T7RNAP from the promoter P B AD vector pBAD18-Cm_t7rnap, bacterial cell cultures were induced with isopropyl β-Dl-thiogalactopyranoside (IPTG) or L-arabinose, depending on the host receiver end of the mentioned plasmids (already either E. coli BL21 or E. coli TOP10, respectively). The detection of the AA peptide by anti-poly-histidine antibodies in western blot assays was carried out using total bacterial extracts, evidencing the conditions in which the T7RNAP could be successfully expressed (FIG 5).

Example 2: Instruction for the cloning of the transposon sequence

 TnC_T7

In order to construct the plasmid that houses the complete transposon sequence TnC_T7 the following steps were performed:

Seq-ID3 was designed to include the R1-R2 binding site for the MuA transposase, a T7 promoter region and the sites for the restriction enzymes EcoRI, BglU, Asc and SamHI (FIG.1). The resulting DNA sequence (Tn_A) was synthesized (Genscript, Piscataway, NJ, USA) and was subsequently cloned into the unique restriction sites EcoRI and phylaHI of plasmid pUC57. The correct orientation of the insertion of Tn_A was verified by digestions with restriction enzymes and / or DNA sequencing. The resulting plasmid is denoted here as pUC57_Tn and is provided in Seq-ID4 (FIG 2).

The kanamycin resistance gene, including its promoter, was amplified by PCR in two independent reactions from the pKD4 plasmid (Datsenko KA, et al., Proc. Nati, Acad. Sci. U. S. A. 2000, 97: 6640-5). In order to replace the BglU restriction site in the assembled DNA sequence, the resulting amplified fragments of the kanamycin resistance gene were ligated after enzymatic digestion with Spel. The resulting sequence of the antibiotic resistance gene is provided in Seq-ID5 (the Spel restriction site is underlined).

The 1, 214 bp sequence provided in Seq-ID5 was digested with Asc \ and SamHI, purified and cloned into pUC57_Tn (Seq-ID4), after digestion of the vector with the same restriction enzymes. The correct orientation of the insert was verified in plasmid DNA isolated from resulting bacterial clones by means of restriction enzyme digestion, colony PCR and / or by DNA sequencing of the final construct, which is denoted here as the vector pUC57_Tn_kanAB (FIG 3).

The coding sequence of the T7RNAP and the arabinose-inducible promoter were amplified with Accuzyme as a high fidelity polymerase (Bioline, London, United Kingdom), using DNA purified from the plasmid pBAD18-Cm_t7rnap (FIG.4) as a template and the primers provided in FIG. Seq-ID6 and Seq-ID7 (Asc restriction sites are shown underlined and no additional nucleotides were included in the primer sequences to allow proper digestion of the restriction enzyme on the resulting PCR product). The 3.122 bp amplicon, corresponding to the PBAD_T7RNAP sequence (Seq-ID8), was inserted into the unique Asc restriction site of pUC57_Tn_kanAB (FIG 3), generating the plasmid pUC57_Tn_kanABJ7 (FIG 6). The correct orientation of the insert was verified by digestion with restriction enzymes, colony PCR and / or by DNA sequencing, in plasmid DNA isolated from the resulting bacterial clones transformed with the corresponding ligation reaction.

The cloning of the second end of the transposon, denoted here as Tn_B, was carried out in the vector pUC57_Tn_kanAB_t7 (FIG 6) to generate the plasmid pUC57_TnC_T7 (FIG 7). Tn_B was amplified by PCR with Accuzyme as high fidelity polymerase (Bioline, London, United Kingdom), using the plasmid DNA pUC57_Tn (FIG.2) as template and the primers provided in Seq-ID9 and Seq-ID10 (the sites Hind restriction were shown underlined and 3 additional nucleotides at the 5 'ends of the primers were included to allow adequate enzymatic digestion on the corresponding PCR product). The 155 bp amplicon (Seq-ID1 1) and the plasmid pUC57_Tn_kanAB_t7 were ligated after digestion with the enzyme Hind \\\. The correct orientation of the Tn_B insertion was verified by digestion with restriction enzymes and / or by DNA sequencing.

As a result, the plasmid pUC57_TnC_T7 hosts the transposon TnC_T7, which consequently includes two flanking sites R1-R2 binding for the MuA transposase, two T7 promoter regions, the kanamycin resistance gene and the coding sequence of the T7RNAP under regulation of the promoter P B AD (FIG 8). The final design of the plasmid pUC57_TnC_T7 allows the transposon to be released by restriction with BglW, making it ready for in vitro reactions with the MuA transposase and with any white episomal DNA. Performing this enzymatic restriction with BglW has been shown to be crucial in generating the 5 'protruding nucleotides required for efficient assembly and stability of the Mu transpososome, as well as for performing the strand transfer reactions {Savilahti H, et al .; EMBO J. 1995, 14: 4893-903).

Example 3: Identification of bacterial cells expressing GFP as a result of the transposition of TnC_T7 into plasmid DNA

To evaluate whether the TnC_T7 transposon can increase the expression of genes in episomal DNA, transposition events were carried out with the TnC_T7 transposon in the sensor plasmid pKR-C12 (Riedel K, et al., Microbiology, 2001, 147: 3249- 62), which is unable to express GFP in E. coli because this bacterial host lacks the quorum sensing system necessary for said expression (Riedel K, et al., Microbiology, 2001, 147: 3249-62). Therefore and for purposes of the present invention, the expression of GFP from pKR-C12 in E. coli is only possible if the transcription process starts from any of the T7 promoters provided by TnC_T7.

The purified pKR-C12 plasmid was used as a white episomal DNA for in vitro transposition reactions of TnC_T7 with the MuA transposase enzyme (Thermo Scientific, Waltham, MA, USA), following the manufacturer's recommendations. The resulting reactions were transformed into the bacterial strain E. coli Ep300 (Epicenter-Illumina, Madison, WI, USA), according to standard methods known in the art, using gentamicin and kanamycin as selection markers. The clones of E. coli Epi300 posttransposition of TnC_T7 in pKR-C12 were grown independently in LB medium until they reached an Optical Density (OD) 6 oonm of 0.4 and induced with L-arabinose 0.2% for an additional 5 hours at 30 ° C. The fluorescence detection assays by spectrophotometry were carried out in a Synergy Microplate Reader (BioTek, Winooski, VT, USA). Each crop Bacterial was evaluated in black 96-well polystyrene plates with a light background (Sigma-Aldrich, Saint Louis, MO, USA) and analyzed with an excitation wavelength at 474 nm and emission at 515 nm. As a result of this type of assay, bacterial clones expressing GFP resulting from the transposition of TnC_T7 into pKR-C12 were finally identified (FIG 10). Bacterial clones post-transposition were analyzed to localize the insertion sites of TnC_T7 in pKR-C12, carrying out sequencing analysis by Sanger from primers hybridized in the transposon sequence.

Alternatively, fluorescence detection assays in post-transposing bacterial clones were performed after growing the bacteria at 37 ° C for 14-16 hours on LB-agar plates supplemented with 0.2% L-arabinose and the corresponding selection markers. In this case, GFP expression was analyzed using the IVIS 200 in vivo Imaging System (PerkinElmer, Waltham, MA USA) with the GFP excitation and emission filters and 15 s of luminescence exposure (FIG 1).

Consequently, the TnC_T7 transposon had the ability to initiate the transcription of genes into plasmid DNA and its validation as a genetic tool in an E. coli strain different from BL21 indicated that the expression of T7RNAP occurred from its corresponding gene located within the transposon. The resulting plasmids post-transposition in pKR-C12, obtained from the bacterial clones 1-8 shown in FIG. 1 1, exhibited differential patterns of GFP expression depending on the specific insertion of the TnC TT transposon.

Example 4: Instruction for the cloning of the coding sequence of GFP in a metagenomic context

A fosmid was generated that includes the GFP coding sequence within its metagenomic DNA insert, by cloning said sequence into a single restriction site. For this, the GFP coding sequence (also denoted as gfp) was amplified, including an upstream RBS, with Accuzyme as a high fidelity polymerase (Bioline, London, UK), using purified DNA from plasmid pKR-C12 as template and the initiators provided in Seq-ID12 and Seq-ID13 (Asc restriction sites are underlined and no additional nucleotides were included in the primer sequences to allow adequate restriction enzymatic digestion on the resulting PCR product). The gpp amplicon of 918 bp (Seq-ID14) was introduced into the DNA insert of a metagenomic clone. For example, the purified phosphonic DNA of a metagenomic clone harboring a DNA insert belonging to a soil sample was linearized by enzymatic restriction and used to insert the gfp amplicon described above. Therefore, the original phosphid DNA isolate, denoted here as pCC2FOS_F076 with the restriction enzyme Asc \ and ligated with the gfp amplicon, was digested to generate the F076_GFP (Seq-ID15; FIG. 12) phosphide. The precise orientation of the insert was verified by digestion with restriction enzymes, colony PCR and / or by DNA sequencing of the final construct, from phosphine DNA isolated from the resulting bacterial clones transformed with the corresponding ligation reaction.

Example 5: Identification of bacterial cells expressing GFP as a result of the transposition of TnC_T7 into phosphonic DNA

To evaluate the ability of the TnC_T7 transposon to increase the expression of genes in phosphine DNA, transposition events were carried out in the F076_GFP fosmid (FIG 12). Therefore, the purified F076_GFP phosphide was used as white episomal DNA for in vitro transposition reactions of TnC_T7 with the MuA transposase enzyme (Thermo Scientific, Waltham, MA, USA), following the manufacturer's recommendations. The resulting reactions were transformed into the E. coli Epi300 bacterial strain (Illumina Inc., San Diego, CA, USA), according to standard methods known in the art, using kanamycin as a selection marker.

The fluorescence detection assays in posttransposition bacterial clones in F076_GFP were carried out after growing the bacteria at 37 ° C for 14-16 hours in dishes of LB-agar supplemented with 0.2% L-arabinose and the corresponding selection marker . GFP expression was evaluated using the IVIS 200 in vivo Imaging System (PerkinElmer, Waltham, MA USA) with GFP excitation and emission filters and 15 s of luminescence exposure (FIG 13). Sanger sequencing analyzes were carried out from the hybridization primers on the transposon sequence, to identify the insertion sites of TnC_T7 in F076_GFP, from the resulting bacterial clones post-transposition. Consequently, the validation in the use of the transposon TnC_T7 to initiate the gene transcription in fosmídico DNA was achieved following the procedures described here.

Example 6: Identification of bacterial cells expressing lipolytic activity as a result of the transposition of TnC_T7

The increase in other enzymatic activities, different from GFP expression, was evaluated in clones derived from metagenomics using the TnC_T7 transposon.

For example, the phosphid vector pCC2FOS_14gF2 isolated from a metagenomic library constructed with DNA derived from soil was used to detect lipolytic activity, since previously a potential active site of Iipase (InterProScan: IPR002168) had been identified by in silico analysis on the insert of the Metagenomic DNA sequenced.

The fosmido pCC2FOS_14gF2 was used as a white episomal DNA for in vitro transposition reactions of TnC_T7, as described in examples 3 and 5, since the previous functional tests to evaluate the degradation of tributyrin in LB-agar medium or degradation of -nitrophenyl butyrate (Sigma-Aldrich, Saint Louis, MO, USA) of the metagenomic clone (hosting pCC2FOS_14gF2) did not show significant differences compared to the baseline for the negative control of lipolytic activity (E. coli Epi300 pCC2FOS).

The transposition reactions of TnC_T7 on pCC2FOS_14gF2 were transformed in the bacterial strain E. coli Epi300 and selected with chloramphenicol and kanamycin. Post-transposition clones were grown independently in LB medium until they reached an OD 6 of 0.4 and induced with 0.2% L-arabinose for an additional 5 hours at 37 ° C. The resulting bacterial cultures were normalized by OD and their respective pellets washed with Tris-HCl buffer solution and re-suspended in 1/5 of its original volume in Tris buffer. Complete bacterial extracts were obtained after lysis of cells using a Mini-Beadbeater-96 (Biospec Products, Bartlesville, OK, USA) and purification by filtration. Following the described methods, the functional assays with a set of E. coli Epi300 pCC2FOS_14gF2 post-transposition clones showed significant increases in lipolytic activity by the degradation of 4-nitrophenyl butyrate (FIG. 1), by quantifying by absorbance at 410 nm in a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA) (FIG 14).

Claims

1. A transposon to partially supply the transcriptional machinery of the host during functional analyzes of genomic / metagenomic libraries, the transposon characterized because it comprises:
(i) a selection marker different from that which is located in the skeleton vector,
(ii) a gene encoding the T7RNA polymerase under an inducible promoter,
(iii) two T7 flanking promoters reading to the outside,
(iv) two inverted repeat recognition flanking sites for the MuA transposase.
2. The transposon according to claim 1, wherein the selection marker different from that which is located in the skeleton vector is the kanamycin resistance gene, including both its promoter and its transcriptional termination signal.
3. The transposon according to claim 1, wherein the promoter inducible for the gene encoding the T7RNA polymerase is the arabinose inducible promoter (PBAD) -
4. The transposon according to claim 1, wherein each of the two inverted repeat recognition flanking sites for the MuA transposase is adjacent to an individual T7 promoter reading to the outside.
5. An artificial vector containing the transposon of claims 1 to 4, characterized in that said vector is the result of the specific combination of the following DNA elements:
(i) a skeleton vector with a high copy number origin of replication and a selection marker,
(ii) a selection marker different from that which is located in the skeleton vector, (iii) a gene that encodes the T7RNA polymerase under an implantable promoter,
(iv) two prompter flanking sequences 17 of reading outwards,
(v) two inverted repeat recognition flanking sites for the MuA transposase.
6. The artificial vector according to claim 5, which has a skeletal vector with the origin of replication ColE1 / pMB1 / pBR322 / pUC and an ampicillin resistance gene as selection marker.
7. The artificial vector according to claim 5, wherein the selection marker different from that which is located in the skeletal vector is the kanamycin resistance gene, including both its promoter and its transcriptional termination signal.
8. The artificial vector according to claim 5, wherein the promoter inducible for the gene encoding the T7RNA polymerase is the arabinose inducible promoter (PBAD) -
9. The artificial vector according to claim 5, wherein each of the two inverted repeat recognition sites for the MuA transposase is adjacent to an individual T7 promoter reading to the outside.
10. The artificial vector according to claims 5 to 9, wherein said vector can be maintained in a bacterial host cell.
1 1. An artificial DNA sequence for locating a MuA binding site and a promoter 17 on different strands of DNA, characterized in that it comprises:
(i) a promoter sequence 17,
(ii) an inverted repeat recognition site for the transposase
MuA, (Ü) multiple flanking recognition sites for restriction endonucleases.
12. The artificial DNA sequence of claim 1, wherein said MuA binding site corresponds to an inverted repeat recognition site for the transposase.
13. The artificial DNA sequence of claim 1, which includes the T7 promoter sequence for specific interaction with T7RNAP and the following recognition flanking sites for restriction endonucleases: EcoRI, BglW, Asc and SamHI.
14. An artificial vector resulting from the specific combination of the following DNA elements:
(i) a skeleton vector with a high copy number origin of replication and a selection marker,
(ii) a T7 promoter sequence with a specific orientation, and
(iii) an inverted repeat recognition site for the MuA transposase in a specific orientation.
15. The artificial vector of claim 14, which has which has a skeletal vector with the ColE1 / pMB1 / pBR322 / pUC origin of replication and an ampicillin resistance gene for selection after transformation into the bacterial host.
16. An artificial vector resulting from the specific combination of the following DNA elements:
(i) a skeleton vector with a high copy number origin of replication and a selection marker,
(ii) a T7 promoter sequence reading to the outside,
(iii) an inverted repeat recognition site for the transposase
MuA, (iv) a selection marker different from that which is located in the skeleton vector.
17. The artificial vector of claim 16, wherein the selection marker different from that which is located on the skeletal vector is the kanamycin resistance gene, including both its promoter and its transcriptional termination signal.
18. An artificial vector resulting from the specific combination of the following DNA elements:
(i) a skeleton vector with a high copy number origin of replication and a selection marker,
(I) a gene encoding the T7RNA polymerase regulated by an inducible promoter.
The artificial vector of claim 18, which has a skeletal vector with a ColE1 / pMB1 / pBR322 / pUC origin of replication, a chloramphenicol resistance gene, and the T7RNAP coding sequence cloned at the unique Kpn restriction site \
The artificial vector of claims 18 and 19, wherein the sequence encoding the T7RNAP is located downstream of the arabinose-inducible promoter and upstream of the transcriptional terminators rmB T1 and T2.
21. An artificial vector resulting from the specific combination of the following DNA elements:
(i) a skeleton vector with a high copy number origin of replication and a selection marker,
(ii) a T7 promoter sequence reading to the outside,
(¡I) an inverted repeat recognition site for the transposase
MuA, (iv) a selection marker different from that which is located in the skeleton vector.
(v) a gene encoding the T7RNA polymerase regulated by an inducible promoter.
22. The artificial vector of claim 21, which has a ColE1 / pMB1 / pBR322 / pUC origin of replication, an ampicillin resistance gene and the coding sequence of T7RNAP.
23. The artificial vector of claims 21 and 22, wherein the sequence encoding T7RNAP is located downstream of the arabinose-inducible promoter.
24. The artificial vector of claim 21, wherein the selection marker different from that which is located in the backbone vector is the kanamycin resistance gene, including both its promoter and its transcriptional termination signal.
25. A method for constructing the transposon according to claims 1 to 4, characterized in that it comprises the steps: a) constructing an artificial DNA sequence to locate a MuA binding site and a T7 promoter on different strands of DNA, where the artificial DNA sequence comprises a T7 promoter sequence, an inverted repeat recognition site for the MuA transposase, and multiple recognition flanking sites for restriction endonucleases; b) construct a first plasmid comprising a skeletal vector with the ColE1 / pMB1 / pBR322 / pUC origin of replication and an ampicillin resistance gene for selection after transformation of the bacterial host, and where the plasmid is constructed by cloning of the artificial DNA sequence of step a) in the unique restriction sites EcoRI and SamHI, after treatment with restriction enzymes of both the skeletal vector and the artificial DNA sequence; c) construct a second plasmid comprising the said DNA regions of the first plasmid [step b)], plus an additional resistance gene, and wherein said second plasmid is constructed by the cloning of the kanamycin resistance gene, including both its promoter as its transcriptional termination signal, within the restriction sites Asc \ and BamH \ of the first plasmid [step b)]; d) construct a third plasmid, which comprises the ColE1 / pMB1 / pBR322 / pUC origin of replication, a chloramphenicol resistance gene and the coding sequence of T7RNAP, the latter cloned at the unique restriction site Kpn \, and where said third plasmid has the coding sequence of the T7RNAP located downstream of the arabinose inducible promoter and upstream of the rrnB transcriptional terminators T1 and T2; e) constructing a fourth plasmid, as a result of the specific combination of components of the second and third plasmids, and wherein said fourth plasmid results from the cloning of the arabinose inducible promoter and the sequence encoding the T7RNAP at the unique restriction site Asc. of the second plasmid between the artificial sequence of step a) and the DNA sequence of kanamycin resistance in the artificial vector; f) constructing a fifth plasmid, which comprises all the structural elements of the fourth plasmid plus a second end of the transposon, where said second end of the transposon is specifically cloned into the unique restriction site H¡nd \\\\ and g) releasing the transposon through the treatment of the restriction enzyme fíg / ll on the fifth plasmid.
26. An artificial vector resulting from the specific combination of the following DNA elements:
(i) a skeletal fosmoid with low to high copy number origins of replication and a selection marker, (ü) a metagenomic DNA insert,
(iii) a gene encoding a variant of the green fluorescent protein (GFP) with a ribosome binding site (RBS) upstream, both specifically located within the metagenomic DNA insert.
27. The artificial vector of claim 26, wherein the phosphid backbone corresponds to the vector pCC2FOS, the metagenomic insert resulted from the random cloning of the metagenomic DNA into pCC2FOS and the reporter gene corresponds to the sequence encoding GFP (including upstream a site of ribosome binding (RBS)), the latter cloned as a single amplicon at the unique Asc restriction site of the metagenomic insert.
28. An artificial vector resulting from the random insertion of the transposon of claims 1 to 4, comprising:
(i) a skeletal vector with its own selection marker and a silenced reporter gene,
(ii) a differential insertion of the transposon of claims 1 to 4 along the DNA sequence of (i).
29. The artificial vector of claim 28, which corresponds to one of the eight plasmids resulting from the random insertions of the TnC_T7 transposon in the pKR-C12 plasmid.
30. A method for increasing DNA transcription as an initial step in the expression of foreign genes, characterized in that it comprises:
(i) generate episomal DNA transposition libraries, as a result of the random insertion of the purified TnC_T7 transposon, where said episomal DNA includes plasmids, fosmides, cosmids or BACs,
(I) introducing said episomal DNA transposition libraries of (i) into host cells,
(iii) expressing the T7RNA polymerase encoded in the TnC_T7 transposon, to generate populations of bacterial host cells with diverse collections of transcripts derived from episomal DNA, (iv) analyzing said populations of bacterial host cells to identify bacterial isolates that express a reporter gene or any other desired function.
PCT/CO2015/000010 2015-05-14 2015-05-14 Novel transposon that promotes the functional expression of genes in episomal dna, and method for increasing dna transcription in the functional analysis of metagenomic libraries WO2016180379A1 (en)

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